Vaccine comprising allogeneic T-cells

ABSTRACT

The invention disclosed herein relates generally to immunotherapy and, more specifically, to the use of immunotherapy for treating tumors and pathogen infected tissues by first priming patients with allogeneic cells designed to be rejected by a Th1 mediated mechanism, then inducing necrosis or apoptosis in a tumor or pathogen infected lesion by methods such as cryotherapy, irreversible electroporation, chemotherapy, radiation therapy, ultrasound therapy, ethanol chemoablation, microwave thermal ablation, radiofrequency energy or a combination thereof applied against at least a portion of the tumor or pathogen infected tissue, and then delivering one or more doses of allogeneic cells (e.g., Th1 cells) within or proximate to the tumor or pathogen-infected tissue in the primed patient. The present invention provides an immunotherapeutic strategy to develop de-novo systemic (adaptive) immunity to a tumor or pathogen.

CROSS-REFERENCE TO RELATED APPLICATION

The present application is a divisional of and claims priority of U.S.patent application Ser. No. 13/150,893, filed Jun. 1, 2011, which is adivisional of and claims priority of U.S. patent application Ser. No.11/936,948, filed Nov. 8, 2007, now U.S. Pat. No. 7,972,594, issued Jul.5, 2011, which is based on and claims the benefit of U.S. provisionalpatent application Ser. No. 60/858,507, filed Nov. 13, 2006, thecontents of which are hereby incorporated by reference in theirentirety.

FIELD OF THE INVENTION

The present invention relates generally to immunotherapy and, morespecifically, to therapeutic methods and compositions for treatingtumors and pathogen infected tissues.

BACKGROUND OF THE INVENTION

Harnessing the power of the immune system to treat chronic infectiousdiseases or cancer is a major goal of immunotherapy. Activeimmunotherapy treatments are methods designed to activate the immunesystem to specifically recognize and destroy tumor or pathogen-infectedcells. For over 200 years active immunotherapy approaches have been usedto prevent numerous infectious diseases, including small pox, rabies,typhoid, cholera, plague, measles, varicella, mumps, poliomyelitis,hepatitis B and the tetanus and diphtheria toxins.

Active immunotherapy concepts are now being applied to developtherapeutic cancer vaccines with the intention of treating existingtumors or preventing tumor recurrence as well as for treatment andprevention of chronic viral infection. Many of these techniques haveproven to successfully develop increased frequencies of immune cells incirculation that have the ability to specifically kill tumors orpathogen infected cells. However, despite the ability to generate immunecells reactive against tumor antigens, tumor escape mechanisms canoverpower this immune response resulting in eventual tumor progression.

Active immunotherapy of cancer has been shown to be very effective innumerous rodent models. However, the clinically disappointing results ofdecades of immunotherapy trials of various types in humans have shownthe immune system in humans does not perceive the threat/danger of humancancer cells as well as the immune system of rodent models of the samediseases.

The same is true of chronic viral infection. The innate immune responseis able to slow down viral replication and activate cytokines whichtrigger the synthesis of antiviral proteins. The adaptive immune systemneutralizes virus particles and destroys infected cells. However,viruses have developed a number of countermeasures to avoid immuneattack and stay moving targets for the immune system.

There is a need to provide an active immunotherapy that is capable ofovercoming tumor and viral immunoavoidance mechanisms and to train thehuman immune system to perceive the threat/danger of human cancer cellsand viral infected cells resulting in an immune response which caneradicate tumors or pathogen-infected cells wherever they might belocated in the body.

SUMMARY OF THE INVENTION

The present invention relates to methods and compositions for inducing asystemic, adaptive immune response against a tumor or pathogen using acombination of an allogeneic cell therapy and a method for subjectingthe tumor or pathogen infected tissue to cellular distress, resulting inthe liberation of tumor specific antigen(s) or pathogen specificantigen(s).

In one aspect of the invention, the present invention is a method forinducing an adaptive immune response against a tumor or a pathogen in asubject. The method includes the steps of: (1) administering to asubject with cancer or an infectious disease an aliquot of allogeneiccells that are designed to be rejected by the subject immune system in amanner that induces anti-allogeneic Th1 immunity; (2) in the samesubject, after allowing time for an anti-allogeneic Th1 immune memory toform (about 7 to 14 days), ablating an accessible tumor lesion orpathogen-infected tissue with a method which causes at least a portionof the tumor or infected tissue to die, preferably by necrosis, (e.g.,by methods such as but not limited to electroporation, cryoablation,chemotherapy, radiation therapy, ultrasound therapy, ethanolchemoablation, microwave thermal ablation, radiofrequency energy or acombination thereof); then; (3) injecting a second aliquot of the sameallogeneic cells intralesionally (same cells as used to prime),preferably 2-24 hrs after the ablation step, creating an immune responsethat serves as an adjuvant to the uptake of antigen(s) and thesubsequent maturation of host antigen presenting cells (i.e., dendriticcells) responding to the necrotic or apoptotic tissue. Mature antigenpresenting cells from the lesion then migrate to the lymph nodes andstimulate systemic anti-tumor or anti-pathogen immunity. In anotheraspect of the invention, the priming step is omitted. The tissue fromthe tumor or the pathogen infected tissue is ablated and an aliquot ofthe allogeneic cells are injected after the ablation to create thedesired immune response.

In another aspect of the invention, the present invention includes amethod of vaccinating a patient. The method includes the steps of: (1)administering to the subject with cancer or an infectious disease apriming composition that includes an aliquot of allogeneic cells thatare designed to be rejected by the subject immune system in a mannerthat induces anti-allogeneic Th1 immunity; (2) in the same subject,after allowing time for an anti-allogeneic Th1 immune memory to develop(about 7 to 14 days), injecting, preferably intradermally, an antigeniccomposition containing a source of tumor antigen or pathogen antigens(e.g., attenuated virus, tumor lysates, heat shock proteins), preferablycontaining from the same individual autologous lysates of the infectedor cancerous tissue, the lysates preferably containing chaperoneproteins, and such lysates formulated with an aliquot of allogeneiccells (same cells used to prime the patient) to create a rejectionresponse and stimulate a delayed-type hypersensitivity response to thealloantigens which serve to adjuvant the stimulation of systemicanti-tumor or anti-pathogen immunity. In a further aspect, this methodmay be practiced without the priming step.

In another aspect, the present invention includes a therapeuticcomposition for treating a tumor or a pathogen in a patient comprisingan antigenic composition that includes tumor antigens or pathogenantigens and an aliquot of allogeneic cells wherein injecting thepatient with the antigenic composition creates an immune responsewhereby subsequent maturation of the patient's antigen presenting cellssystemically stimulate anti-tumor or anti pathogen immunity. The tumorantigens in the composition are derived from necrosis of the tumor. Thepathogen antigens in the composition are derived from necrosis of thepathogen infected tissue. The therapeutic composition may also include apriming composition containing an aliquot of allogeneic cells. Thealiquot of allogeneic cells in the priming composition and in theantigenic composition may include between about 1×10⁸ and about 1×10¹⁰cells.

In another aspect, the present invention includes a vaccine for apatient against a tumor or a pathogen. The vaccine includes an antigeniccomposition comprising antigenic material from the tumor or pathogen andan aliquot of allogeneic cells wherein administration of the antigeniccomposition to the patient creates a rejection response and stimulates adelayed-type hypersensitivity response to the antigens thereby acting asan adjuvant to the stimulation of systemic anti-tumor or anti-pathogenimmunity in the patient. The vaccine can also include a primingcomposition wherein the priming composition includes an aliquot ofallogeneic cells.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

The present invention includes a method for stimulating anti-tumor oranti-pathogen immunity in patients. The method involves first “priming”of the patient to develop Th1 anti-alloantigen immune memory by infusionof an aliquot of allogeneic cells. It is desired that the infusion ofallogeneic cells stimulates the patient's immune system to react againstthe allogeneic cells. A time period is allowed to elapse until thepatient's immune system is allowed to form an anti-allogeneic memory. Insome embodiments, a patient may need a booster of allogeneic cells todevelop the appropriate Th1 immune memory.

Patient as used herein includes not only mice but also humans. Th1response as used herein refers to production of a cytokine profile thatactivates T-cells and macrophages. Th1 response is to be distinguishedfrom Th2 response which activates mainly an immune response that dependsupon antibodies and is antagonistic to the Th1 response.

The next step includes injury and/or death of cells within a tumor bedor pathogen-infected tissue after the patient develops sufficientanti-allogeneic Th1 immune memory. Tissue injury or death releasescellular components and recruits scavenger cells to the injury site. Avariety of methods are known in the art to cause tissue injury or deathwithin a tumor bed or pathogen infected tissue. Preferably death is bynecrosis, which causes recruitment of scavenger cells to the injurysite. In some preferred embodiments, tissue death or injury is bycryoablation or by irreversible electroporation. Alternatively, tissueis ablated ex-vivo and the released components injected into thepatient.

The scavenger cells, including immature dendritic cells can pick upantigens released from the damaged or dead tissues. A second aliquot ofallogeneic cells are injected intralesionally in order to cause thematuration of dendritic cells (DC) for the priming of Th1 immunity tothe antigens. By intralesionally is meant administration of thecomposition of this invention through injection or otherwise directlyinto a cancerous area or tumor or a pathogen infected tissue. Inpreferred embodiments, all of the allogeneic cells administered to thepatient are from the same source. Preferably, the allogeneic cells areadministered between about 2 and about 24 hours after ablation of thetissue. This method is especially useful in the treatment of solid ormetastatic tumors, particularly in patients with tumor lesions residentin the prostate, breast, bone, liver, lung, or kidney.

is desirable for the patient to develop a strong delayed-typehypersensitivity (DTH) reaction upon introduction of the second aliquotof allogeneic cells resulting in rejection of the allogeneic cells dueto the fact that the patient has been primed to Th1 immunity against theallogeneic cells introduced by the first aliquot. The by-stander effectof the anti-alloantigen DTH reaction can produce “danger signals” whichserve to cause the DC, collecting and processing antigens from thedamaged tissue, to mature and migrate to the draining lymph nodes. Thecombination of the induction of tissue injury and the DTH rejectionresponse can create an inflammatory environment which leads to Th1immunity against the antigens released from the damaged tissue.

The general state of inflammation caused by the treatment process canserve to cause the DC to program T-cells to Th1 immunity againstantigens in the damaged tissue resulting in a systemic adaptive immuneresponse to the tumor or pathogen-infected cells and the disabling oftumor and pathogen-mediated immune avoidance mechanisms. By adaptiveimmunity is meant that the patient's defenses are mediated by B and Tcells following exposure to antigen and that such defenses exhibitspecificity, diversity, memory, and self/nonself recognition. Suchadaptive immunity is systemic within the patient. Adaptive immunity isto be distinguished from innate immunity which is non-specific andexists prior to exposure to the antigen.

In some embodiments, ablation followed by administration of theallogeneic cells may be sufficient to generate the desired response. Inother words, the priming of the patient by a first aliquot of allogeneiccells may be omitted. In these embodiments, tissue from the tumor orinfected by a pathogen is ablated followed by injection of an aliquot ofallogeneic cells.

The present invention also includes a method of vaccinating a patienthaving cancerous cells or an infected tissue. This method is,preferably, used for patients with hematological malignancies (e.g.,Chronic Lymphocytic Leukemia, Multiple Myeloma, and non-Hodgkin'slymphomas) or viral infectious diseases (e.g., hepatitis B or C, herpes,HIV) and other disorders where the affected lesions are not easilyassessable for ablation.

The method involves first “priming” of the patient to develop Th1anti-alloantigen immune memory by infusion of a first aliquot ofallogeneic cells. It is desired that the infusion of allogeneic cellsstimulates the patient's immune system to react against the allogeneiccells. A time period is allowed to elapse until the patient's immunesystem is allowed to form an anti-allogeneic memory. In someembodiments, a patient may need a booster of allogeneic cells to developthe appropriate Th1 immune memory.

The next step includes injecting into the patient an antigeniccomposition that includes an autologous lysate containing antigens fromthe cancerous cells or the infected tissue. This composition alsoincludes an aliquot of the allogeneic cells, i.e. allogeneic cells thatare from the same source as the allogeneic cells used in the primingstep. The injection of the antigenic composition can create a rejectionresponse in the patient and can stimulate a delayed-typehypersensitivity response to the antigens.

The scavenger cells, including immature dendritic cells can pick up theantigens from the autologous lysate. The allogeneic cells can cause thematuration of dendritic cells for the priming of Th1 immunity to theantigens. It is desirable for the patient to develop a strong DTHreaction upon introduction of the allogeneic cells with the autologouslysate due to the fact that the patient has been primed to Th1 immunityagainst the allogeneic cells introduced by the first aliquot ofallogeneic cells during the priming step.

The general state of inflammation caused by the treatment process canserve to cause the DC to program T-cells to Th1 immunity against theantigens in the autologous lysate resulting in a systemic adaptiveimmune response to the tumor or pathogen-infected cells and thedisabling of tumor and pathogen-mediated immune avoidance mechanisms.

The present invention also provides a method for enhancing theimmunogenicity of weakly immunogenic or non-immunogenic tumors and amethod to deviate an immune response from a non-protective immuneresponse (e.g., Th2 response) to a protective immune response (e.g.,Th1). Such diseases include, for example, all types of cancers anddiseases caused by infections with a variety of pathogens (e.g.,Hepatitis viruses, fungal infections such as Aspergillus, HIV, malaria,typhoid, cholera, herpes viruses, Chlamydia, and HPV).

The present invention also includes a therapeutic composition fortreating a tumor or a pathogen in a patient. The therapeutic compositionpreferably includes a priming composition and an antigenic composition.The priming composition generally contains allogeneic cells which areinjected into the patient to generate a rejection response by thepatient's immune system in a manner that induces an allogeneic Th1immunity.

The antigenic composition includes antigenic material from the tumor orpathogen-infected tissue and an aliquot of allogeneic cells. Inpreferred embodiments, the antigenic material is an autologous lysatecontaining antigens from the cancerous cells or from infected tissue.The antigenic material can be derived from tissue necrosis of the tumoror the pathogen-infected tissue. Preferably, the antigenic material isderived from ablation of the tumor or pathogen-infected tissue. Theablation may be done in vivo or ex vivo. In some embodiments, theantigenic material includes heat shock proteins released upon ablationof the tissue from a tumor or pathogen-infected tissue.

The antigenic composition also includes allogeneic cells. The antigenicmaterial and the allogeneic cells may be combined together or packagedseparately. The antigenic composition including the antigenic materialand the allogeneic cells, when injected into the patient, can create arejection response and stimulate a delayed-type hypersensitivityresponse to the antigens thereby acting as an adjuvant to thestimulation of systemic anti-tumor or anti-pathogen immunity in thepatient.

The therapeutic compositions may include other components that act asadjuvants to the response generated by the priming composition and theantigenic composition. The priming composition and antigenic compositionmay include other components generally found in therapeutic composition,for example, preservatives. The addition of these components are withinthe scope of this invention.

In some embodiments, the therapeutic composition may only include theantigenic composition and not the priming composition. The antigeniccomposition may be sufficient to obtain the desired immune response.

The present invention also includes a vaccine for a patient against atumor or a pathogen. The vaccine preferably includes a primingcomposition and an antigenic composition. The priming compositiongenerally contains allogeneic cells which are injected into the patientto generate a rejection response by the patient's immune system in amanner that induces an allogeneic Th1 immunity.

The antigenic composition includes antigenic material from the tumor orpathogen-infected tissue and an aliquot of allogeneic cells. Inpreferred embodiments, the antigenic material is an autologous lysatecontaining antigens from the cancerous cells or from infected tissue.The antigenic material can be derived from tissue necrosis of the tumoror the pathogen-infected tissue. Preferably, the antigenic material isderived from ablation of the tumor or pathogen-infected tissue. Theablation may be done in vivo or ex vivo. In some embodiments, theantigenic material includes heat shock proteins released upon ablationof the tissue from a tumor or pathogen-infected tissue.

The antigenic composition also includes allogeneic cells. The antigenicmaterial and the allogeneic cells may be combined together or packagedseparately. The antigenic composition including the antigenic materialand the allogeneic cells, when injected into the patient, can create arejection response and stimulate a delayed-type hypersensitivityresponse to the antigens thereby acting as an adjuvant to thestimulation of systemic anti-tumor or anti-pathogen immunity in thepatient.

The vaccine may include other components that act as adjuvants to theresponse generated by the priming composition and the antigeniccomposition. The priming composition and antigenic composition mayinclude other components generally found in vaccines, for example,preservatives. The addition of these components are all within the scopeof this invention.

In some embodiments, the vaccine may only include the antigeniccomposition and not the priming composition. The antigenic compositionmay be sufficient to obtain the desired immune response.

The therapeutic vaccines of the present invention are useful for theprevention and treatment of diseases such as cancer or chronic viraldisease which develop and/or persist by suppressing or escaping theimmune response.

Priming Step

The purpose of the priming step is to create anti-allogeneic Th1immunity in a patient that can be recalled upon subsequent exposure tothe alloantigens. Priming occurs by exposing a patient to an aliquot ofallogeneic cells and the subsequent rejection of these allogeneic cellswhen a second aliquot is administered to the patient by the patient'simmune system resulting from immune memory. Preferably, the patients arenot immunosuppressed prior to priming, as this will inhibit the abilityof the patient to reject the infused allogeneic cells and will alsoinhibit the development of anti-alloantigen Th1 immunity.

In one embodiment of the present invention, the patient's immune systemis skewed to generate Th1 immunity. It is preferable to manipulate theallogeneic cells such that Th1 and not Th2 immunity develops in responseto the rejection of the allogeneic cells. In one embodiment, thepatient's immune system can be skewed to produce Th-1 response byadministering allogeneic cells that are producing Th1 cytokines (e.g.,IFN-gamma and TNF-alpha) when infused. Th1 cytokines can assist inskewing the immune response to the alloantigens to Th1 type immunity.Other methods of skewing a patient's immune system to produce Th-1immunity are also within the scope of this invention.

The allogeneic cells used to first prime the patients and then laterused for either intralesional administration (after induction of celldeath) or as an adjuvant to a source of pathogenic or tumor material,are preferably allogeneic activated T-cells, more preferably allogeneicactivated CD4+Th1 cells, more preferably allogeneic CD4+ T-cells thathave differentiated into effector or memory cells and produce highlevels of Type 1 cytokines, such as IL-2, IL-15, IFN-gamma, TNF-alphaand also express, preferably at high density, effector molecules such asCD40L, TRAIL and FasL on the cell surface but do not produce IL-4 orother Type 2 cytokines. CD40 ligation of innate immune cells (e.g.,dendritic cells, macrophages and NK cells) has the capacity to inducehigh levels of the cytokine IL-12, which polarizes CD4+ T cells towardthe Th1 type immunity, enhances proliferation of CD8+ T cells, andactivates NK cells. These pro-inflammatory events can enable theconsistent development of Th1 immunity to the alloantigens on theallogeneic cells upon rejection by the patient's immune system.

In the priming step, the activated allogeneic T-cells are administeredto the patient, preferably intravenously, but can also be administeredintradermally. The allogeneic cells are preferably derived from adeliberately HLA-mismatched donor. Preferred dosage in an aliquot ofallogeneic cells for intravenous infusion is at least about 1×10⁷ cellsand more preferred is between about 1×10⁸ to 1×10¹⁰ cells. Dosages ofallogeneic cells outside this range that can primarily generate animmune response are also within the scope of this invention.

It is desirable to test the patients for development of Th1anti-alloantigen immunity prior to the ablation of affected tissue oradministration of the antigenic composition. The development of Th1anti-alloantigen immunity may take at least about 7 days. Preferably,the patient is allowed between about 7 days to about 14 days to developTh1 anti-alloantigen immunity. The development of Th1 anti-alloantigenimmunity can be measured by, for example, ELISPOT assay. Other methodsof testing patients for development of Th1 anti-alloantigen immunity arealso within the scope of this invention. If the Th1 anti-alloantigenimmunity is weak, additional booster injections of allogeneic cells canbe administered. Booster injections are preferably made intradermally togenerate a delayed type hypersensitivity (DTH) reaction in the skin.

Generation of Allogeneic T-Cells

It is desirable that allogeneic T-cells can be generated such that,upon, activation and infusion into a patient, a Th-1 immunity can begenerated by the patient. A preferred method for producing allogeneiccells with the properties necessary for stimulation of anti-allogeneicTh1 immunity involves: (1) the collection of mononuclear cell sourcematerial by leukapheresis from normal screened donors; (2) the isolationof CD4 T-cells from the source material; (3) the activation of the CD4+cells with immobilized anti-CD3 and anti-CD28 monoclonal antibodies(mAbs) on days 0, 3 and 6; (4) the activation of the cells again on day9 with immobilized anti-CD3 and anti-CD28 mAbs and the infusion of thecells within 24 h of activation.

Cell Death Step

Cell death or cell injury can result in recruitment of DC to the lesionand provide a source of antigen for uptake by DC. It is preferable thattarget tissues be destroyed by a process which causes death by necrosis.By necrosis it is meant the death of individual cells or groups of cellssuch that amounts of intracellular components are released to theenvironment. For purposes of this application, necrosis includes a celldeath by a variety of methods including cryoablation, irreversibleelectroporation, chemotherapy, radiation therapy, ultrasound therapy,ethanol chemoablation, microwave thermal ablation, radio frequencyenergy or a combination thereof. Necrotically killed cells activateendogenous signals of distress responsible for the recruitment andmaturation of DC, stimuli that would not be generated by healthy orapoptotically dying cells. Further, exposure of immature DC to thesestimuli provides maturation signals, critical for the initiation oflocal and systemic Th1 immunity.

In one preferred embodiment, in order to cause death by necrosis, it ispreferred that the target tissue is frozen. Cryosurgery is a well-aimedand controlled procedure capable of inducing tissular necrosis by theapplication of liquid N2 or argon gas. The biologic changes that occurduring and after cryosurgery have been studied in vitro and in vivo.Tissue injury and necrosis is induced by cell freezing and by thevascular stasis that develops after thawing. Cryosurgery (in situfreezing) has been known to elicit an antigenic stimulus (comparable tothat obtained through the parenteral administration of antigen) capableof generating a specific immunologic response against autologousantigens of the frozen tissue.

Cryoablation can cause peptides to be released from lysed tumor orpathogen-infected cells for antigen processing by DC and creates apro-inflammatory cytokine environment. Cytokines released aftercryoablation such as IL-1, IL-2, TNF-α, IFN-γ, and GM-CSF can activatethe T, NK, and Langerhans cells essential to an immune response capableof destroying cancer or pathogen infected cells.

In another preferred embodiment, in order to cause death by necrosis, itis preferred that the target tissue is subject to irreversibleelectroporation. Irreversible electroporation is a tissue ablationtechnique in which micro to milli-second electrical pulses are deliveredto the tissue to produce cell necrosis through irreversible cellmembrane permeabilization. In irreversible electroporation, the cellularmembranes of the cells between the electrodes are disrupted causingcellular necrosis. Irreversible electroporation can cause antigens to bereleased from lysed tumor or pathogen-infected cells for antigenprocessing by DC and creates a pro-inflammatory cytokine environment.

Another preferred method for generating a source of antigen is toisolate autologous chaperone proteins, also known as heat shock proteins(HSP), from dead infected tissue or tumors. HSPs are among the majortargets of the immune response to bacterial, fungal and parasiticpathogens. Certain chaperones in extracellular milieu may also modulateinnate and adaptive immunity due to their ability to chaperonepolypeptides and to interact with the host's immune system, particularlyprofessional antigen-presenting cells. Vaccination with heat shockproteins from tumor have been shown to elicit an anti-tumor response.Current studies indicate that the immunogenicity of HSPs is derived fromthe antigenic peptides with which they associate.

A preferred method for isolation of chaperone proteins for use as anantigen source is described by Katsantis in U.S. Pat. No. 6,875,849.Additional methods are described by Srivastava in U.S. Pat. Nos.6,797,480; 6,187,312, 6,162,436; 6,139,841; 6,136,315; and 5,837,251.

Adjuvant Step

The purpose of the adjuvant step is to cause the maturation of DC tostimulate Th1 immunity against antigens taken up in the lesionscontaining dead target tissue. This can be accomplished by the injectionof the same allogeneic cells, i.e. allogeneic cells of the same originas those used to prime the patient. This aliquot of the allogeneic cellsare, preferably, injected intralesionally, i.e. directly into thenecrotic lesion caused by the cryoablation, or other method of celldeath. Alternatively, when chaperone proteins are used as the source ofantigen, the same allogeneic cells used to prime the patient areinjected with the chaperone proteins, preferably intradermally. Thedosage of the allogeneic cells to generate the desired immune responseis generally at least about 1×10⁷ cells and more preferred is betweenabout 1×10⁸ to 1×10¹⁰ cells. Dosages of allogeneic cells outside thisrange that can generate the desired immune response are also within thescope of this invention. The preparation of the allogeneic cells is thesame as described above.

To initiate an immune response and overcome the natural tolerance theimmune system has to self tissues, the antigens released after necroticcell death or associated with the chaperone proteins must be taken up byDC and presented with immune activating components that signal “danger”.The memory immune response against the allogeneic cells create this“danger”.

The tissue resident DC, termed immature DC, are able to capture Ag fromthe environment, but are deficient in stimulating T cells. In responseto pathogen infection and the ensuing inflammatory response, DC undergoa differentiation process called maturation, whereby they up-regulatethe capacity to migrate to draining lymph nodes and present the capturedantigens to T cells. To activate Th1 CD4⁺ T cells and CTL, the DC has tointegrate a number of maturation/differentiation stimuli. At the site ofpathogen or tumor encounter, exposure to pathogen or tumor-deriveddeterminants, proinflammatory cytokines, and/or cell debris induces thefirst steps in the maturation process. This includes the up-regulationof costimulatory molecules and chemokine receptors, whereby the DCacquire the ability to present antigens to T cells and migrate to thelymph node, respectively. At the lymph node, encounter of cognate CD4⁺ Tcells provides additional differentiation stimuli to the DC, whichregulate the survival of the activated T cells and the polarization ofthe CD4⁺ T cells.

The maturation of DC occurs at the site of antigen uptake and the recallrejection response serves as an adjuvant to provide the appropriateinflammatory danger signals necessary for DC maturation, migration tothe lymph nodes and the programming for Th1 immunity against theantigens uptaken in the lesion.

EXAMPLES

Animals

Balb/c mice were hosts and C57B1/6 (B6) mice were used as source of Th1cells. All mice were 6 to 10 weeks old, were maintained in a specificpathogen-free facility at the Hadassah-Hebrew University Medical Center,and were treated on an approved animal protocol.

Preparation of Allogeneic Th1 Memory Cells

Spleen cells from male C57BL/6 mice were harvested and treated withammonium chloride-potassium (ACK) buffer for lysis of red blood cells.Approximately 70-100 million cells were isolated per spleen. CD4+T-cells were then purified by positive selection (purity >98%) using CD4immunomagnetic particles on an MS column (Miltenyi Biotec, Germany),approximately 8-12 million CD4 cells were isolated with a yield of50-60%. Th1 memory cells were generated by expansion with anti-CD3 andanti-CD28-coated paramagnetic beads (CD3/CD28 T-cell expander beads,Dynal/Invitrogen) at an initial bead:CD4 cell ratio of 3:1. The purifiedCD4 cells were incubated with 20 IU/mL recombinant mouse (rm)IL-2, 20ng/mL rmIL-7, and 10 ng/mL rmIL-12 (Peprotech, New Jersey) and 10 μg/mLantimurine IL-4 mAb (Becton Dickenson) in RPMI 1640 media containing 10%FBS, penicillin-streptomycin-glutamine, nonessential amino acids (NEAA)(Biological Industries, Israel) and 3.3 mM N-acetyl-cysteine (NAC;Sigma) (complete media). Additional cytokine-containing complete mediawith rmIL-2 and rmIL-7 was added to the CD4 cultures daily from days 3to 6 to maintain the cell concentration between 0.5 and 1×10⁶ cells/mL.Additional CD3/CD28 beads were added daily from day 3 to day 6. Thenumber of beads added was calculated to maintain a 1:1 bead:cell ratioas the cells expanded. After 6 days in culture, the CD4 cells expandedapproximately 80 to 100-fold and were harvested and debeaded by physicaldisruption and passage over a magnet. The phenotype of the harvestedcells used in experiments were >95% CD4+, CD45RO+, CD62L^(lo), IFN-α+and IL-4−.

CD3/CD28 Nanobead Preparation

Biotinylated mouse anti-CD3 and anti-CD28 mAbs (BD Pharmingen) were eachdiluted in 400 μl of PBS to a final concentration of 25 μg/ml and thenmixed in a 1:1 ratio so that the final volume was 800 μl. 20 μl ofStrepavidin-coated nanobeads (Miltenyi, Germany) were washed and dilutedto a final volume of 200 μl in PBS. The 800 μl of the CD3/CD28 mAbsolution and the 200 μl of diluted nanobeads were then mixed so that thefinal concentration of each mAb was 10 μg/ml in a total volume of 1 ml.The mixture was placed on a rotating mixing device for 30 min at RT. ThemAb conjugated nanobeads were then passed over an MS column (Miltenyi,Germany) on a magnet and washed thoroughly. The retained nanobeads werethen released from the column and resuspended in 200 μl of PBS. Thenanobeads were not able to activate naïve T-cells. Therefore, thenanobeads were tittered against harvested Th1 memory cells that had beenprevious activated 6 days prior with CD3/CD28 T-cell expander beads(Dynal, Norway). While there were slight variations per batch, generally20 μl/10⁷ cells was found to provide optimal activation of previouslyactivated Th1 memory cells.

CD3/CD28 Cross-Linking

In experiments that required the infusion of activated Th1 memory cells,the harvested Th1 cells were incubated with a pre-tittered concentrationof CD3/CD28-conjugated nanobeads prior to infusion. For optimalactivation, the cells had to be incubated with the nanobeads for aminimum of 4 h and a maximum of 18 h. Optimal activation causedproduction of IFN-γ and upregulation of CD40L and FasL on the cellsurface. For these experiments, all infusions of CD3/CD28 cross-linkedTh1 memory cells occurred after 4-8 h of pre-incubation. Cells werethoroughly washed prior to infusion to remove any unassociatednanobeads. Cross-linked Th1 memory cells used in these experimentsexpressed FasL and CD40L on the cell surface and produced in excess of2000 ng/ml/10⁶ cells/6 h IFN-γ and less than 20 μg/ml IL-4 per 10⁶cells/6 h. Th1 memory cells without CD3/CD28 cross-linking did notproduce cytokines or express FasL or CD40L.

Cryotherapy

Cryotherapy was performed with a spherical nitrous oxide cryoprobe, 3 mmin diameter. The gas was maintained at a pressure of 50 bars and theJoule-Thomson effect allowed to attain temperatures ranging from −30 to−40° C. in the tissue. An incision was made in the centre of the tumor,the cryoprobe was placed in contact with the tumor (it was inserted 1-2mm deep): the aim was to influence it by freezing but not to destroy itcompletely. Three cycles of rapid freezing (lasting for 20 s) followedby slow thawing were applied. The ice ball was produced at the center ofthe lesion and reached about two thirds of the total tumor volume.

Example #1

To test the ability of allogeneic Th1 cells to stimulate systemicanti-tumor immunity in extensive metastatic disease, the followingprotocol was tested. Lethal doses of tumor cells including BCL1leukemia, 4T1 breast cancer and 3LL lung cancer were infusedintravenously into mice on day 0 and the tumor cells were also injectedintradermally to establish a solid tumor mass. On day 7, the mice weregiven a 1×10⁵ dose of allogeneic Th1 cells. On day 14, the mice weretreated intratumorally by injection of either: (a) saline; (b)saline+partial cryoablation of tumor; (c) allogeneic Th1 cells at a doseof 10³ cells; or (d) allogeneic Th1 cells+partial cryoablation of tumor.The results of surviving animals at 90 days is shown below (n=10):

Intratumoral BCL1 4T1 3LL treatment Leukemia Breast Lung Saline 0 (0%) 0(0%) 0 (0%) Saline + cryoablation 0 (0%) 0 (0%) 0 (0%) Th1 alone 1 (10%)1 (10%) 2 (20%) Th1 + cryoablation 4 (40%) 5 (50%) 8 (80%)

Example #2

In order to investigate whether treatment of patients with solid tumorsmight benefit from the present invention, the experiment design abovewas repeated in animals that only received intradermal injections oftumors creating solid tumor masses. The results were similar to thoseobtained with animals with metastatic disease.

Intratumoral BCL1 4T1 3LL treatment Leukemia Breast Lung Saline 0 (0%) 0(0%) 2 (20%) Th1 alone 0 (0%) 0 (0%) 1 (10%) saline + cryoablation 0(0%) 0 (0%) 2 (20%) Th1 + cryoablation 6 (60%) 7 (70%) 9 (90%)

The combination of Th1 cells with cryotherapy results in high curerates. Cryotherapy kills tumors by necrosis, which is thought to be amore pathological type of cell death than death by apoptosis (the typeof death caused by chemotherapy). It is thought that the cryotherapymakes the tumors more immunogenic and therefore the combination ofallogeneic Th1 cells with necrotic tumor death creates a type of tumorvaccine leading to systemic anti-tumor immunity.

Although the present invention has been described with reference topreferred embodiments, workers skilled in the art will recognize thatchanges may be made in form and detail without departing from the spiritand scope of the invention.

What is claimed is:
 1. A therapeutic composition for treating a pathogenin a patient comprising: pathogen antigens wherein the pathogen antigensare pathogen infected tissue subjected to necrosis and wherein thepathogen antigens comprise chaperone proteins; and allogeneic activatedT-cells associated with nanobeads suspended in media suitable forinjection to the patient, wherein the allogeneic T-cells serve as anadjuvant to create an immune response, whereby the antigen-presentingcells in the patient uptake the antigens and subsequently mature in thepatient to systemically stimulate anti-pathogen immunity, wherein theallogeneic cells are CD4+Th1 cells producing in excess of 2000 ng/ml/10⁶cells/6 hours of IFN-γ, wherein the CD4+Th1 cells were produced bynormal donor T-cells expanded by CD3/CD28 T-cell expander beads,harvested after expansion, debeaded, and the debeaded cells incubatedfor about 4 to about 18 hours with nanobeads conjugated to antibodiesthat bind to CD3 and CD28 cell surface moieties for cross-linking ofCD3/CD28 on the T-cells prior to infusion.
 2. The composition of claim 1further comprising a priming composition wherein the priming compositioncomprises allogeneic cells.
 3. The composition of claim 1 wherein thepathogen antigens are generated in vivo.
 4. The composition of claim 1wherein the pathogen antigens are generated ex vivo.
 5. The compositionof claim 1 wherein the activated T-cells are producing Th1 cytokines. 6.The composition of claim 1 wherein the composition comprises betweenabout 1×10⁷ and about 3×10⁷ cells.
 7. The composition of claim 1 whereinthe activated T-cells are not pathogen specific T-cells.
 8. A vaccinefor a patient against a pathogen comprising: antigenic material fromtissue infected by the pathogen, wherein the antigenic materialcomprises chaperone proteins; and allogeneic activated T-cellsassociated with nanobeads suspended in media suitable for injection,wherein the allogeneic cells create a rejection response and stimulate adelayed-type hypersensitivity response in the patient to the allogeneiccells thereby acting as an adjuvant to the stimulation of systemicanti-pathogen immunity in the patient, wherein the antigenic material isgenerated by necrosis of a lesion and wherein the allogeneic cells areCD4+Th1 cells producing in excess of 2000 ng/ml/10⁶ cells/6 hours ofIFN-γ, wherein the CD4+Th1 cells were produced by normal donor T-cellsexpanded by CD3/CD28 T-cell expander beads, harvested after expansion,debeaded after expansion, and the debeaded cells incubated for about 4to about 18 hours with nanobeads conjugated to antibodies that bind toCD3 and CD28 cell surface moieties for cross-linking of CD3/CD28 on theT-cells prior to infusion.
 9. The vaccine of claim 8 further comprisinga priming composition wherein the priming composition comprisesallogeneic cells.
 10. The vaccine of claim 8 wherein the pathogenantigens are generated in vivo.
 11. The vaccine of claim 8 wherein thepathogen antigens are generated ex vivo.
 12. The vaccine of claim 8wherein the activated T-cells are producing Th1 cytokines.
 13. Thevaccine of claim 8 wherein the vaccine comprises between about 1×10⁷ andabout 3×10⁷ cells.
 14. The vaccine of claim 8 wherein the activatedT-cells are not pathogen specific T-cells.